Background Skeletal muscle is similar to nerve tissue in that the fiber responds to a stimulus in an all-or-none fashion.

This response is called a twitch. In living organisms, skeletal muscles are stimulated by motor neurons. A single motor neuron, and all the muscle fibers that it innervates, is known as a motor unit (Figure 1).

Figure 1 the constituent parts of a motor unit (Campbell et al. 2009).

Motor units vary greatly in size, from just a few muscle fibers innervated by a single neuron (small motor unit), up to thousands of muscle fibers per neuron (large motor unit). The smaller the motor unit, the finer the control of movement in that muscle. Thus the muscles controlling the movements of the fingers and eyes have small motor units, whereas those controlling the large limb muscles may have very large motor units. However, most muscle consists of a number of motor units, covering a range of sizes. Depending on the intensity and frequency of stimulation, greater numbers of muscle fibers are activated. The strength of a muscle contraction, therefore, can be increased in two ways: by increasing the number of active motor units (termed recruitment), and by stimulating existing active motor units more frequently (mechanical summation). The absolute force that a muscle can generate is dependent on the size and total number of muscle fibers. So muscles with large cross-sectional areas are able to generate larger forces than those with small cross-sectional areas. In your experimental setup there will be 3 sources of delay between delivering the electrical stimulus to the sciatic nerve and contraction of the gastrocnemius muscle. The first source of delay is the time taken for the compound action potential to propagate down the length of the sciatic nerve; this was investigated in your previous practical class on action potentials (Kibedi and Theiss 2010).

The second source of delay is at the junction between a motor nerve and a muscle fiber. Here there is a specialised type of synapse called the neuromuscular junction (easily remembered as it is the junction between the neuron and the muscle) (Figure 2). Motor nerves release the neurotransmitter acetylcholine from their terminals (Hudson et al. 2005). The acetylcholine released into the junction cleft binds to receptors on the muscle membrane that are directly coupled to selective ion channels.

Figure 2 - a neuromuscular junction and excitation-contraction coupling (Campbell et al. 2009).

Opening of these channels depolarizes the muscle membrane, and leads to the generation of an action potential which spreads across the muscle membrane. This muscle action potential propagates across the muscle membrane and down the T tubules. This in turn causes the release of intracellular Ca2+ from the sarcoplasmic reticulum. The increased cytosolic calcium binds to the troponin-tropomyosin complex and sets in motion the biochemical events that cause contraction (Bennett 2010). All the processes which occur between the generation of the muscle action potential and contraction are collectively called excitation-contraction coupling; this is the third source of delay in your setup.

Skeletal muscle can be studied under isometric (constant length) or isotonic (constant load) conditions. Here, as in your experiments, the force is measured isometrically. Action potentials in skeletal muscle, like those in nerve, last for only a few milliseconds. In contrast, the mechanical response of the muscle - the contraction lasts significantly longer (Figure 3).

Active force

Passive force

Figure 3 an example recording of total force (red) generated by a skeletal muscle contraction. Also shown are the contributions of passive (green) and active (blue) forces to the total.

A second stimulus arriving before the muscle has relaxed again, causes a second twitch on top of the first, so that greater total force developed. This is called mechanical summation. With increasing frequency of stimulation, there is less and less time for the muscle fibers to relax between stimuli and eventually the contractions fuse into a smooth powerful contraction - tetanus. The active force of muscle contraction is also influenced by the passive force. The more a muscle is stretched, the more tension (passive force) it is under. In considering the force of the muscle in response to stimulation, it is necessary to separate out the passive and active forces (Figure 3). The passive forces reflect the contributions of elastic elements in the muscle; the active force is that generated by the contractile elements when the fibers are stimulated. Experimentally, what is measured at different degrees of stretch is the total force. The passive force can be measured prior to stimulation, and the difference between this and the total force is the active force, at any given muscle length. Skeletal muscle contraction requires metabolic energy. A depletion of energy stores results in fatigue. This can be counteracted to some extent by ensuring the preparation is frequently washed with fresh Ringers solution; nevertheless, it is an important consideration when designing your experiments that you do not fatigue the tissue.

Experimental design: Now that you have a good understanding of the background material, you can proceed to design your own experiment. In the practical class, your experimental set up will allow you to deliver multiple electric stimuli to the toad sciatic nerve. You can alter the strength, duration, and frequency of stimuli. You will all do a brief preliminary investigation to analyse the effect of altering stimulus strength on the peak contractile force generated by the gastrocnemius muscle. You will then be asked to complete a further experiment of your own design. You should have a specific hypothesis and an outline of your experiment before you come to class, this will enable you to discuss materials and methods with your group members and tutor at the start of the class. The following equipment will be available for your use during the practical class. You should base your hypothesis and materials and methods on what is achievable in the 3-hour time frame (it is better to have time spare at the end). The equipment provides a range of different methods which can be used. You are not expected to use all of the equipment provided. Sciatic nerve and gastrocnemius muscle of a cane toad, Bufo marinus. Powerlab and stimulating electrode can vary strength, duration, and frequency of stimulus. Force transducer measures the amount of force produced by the muscle. Clamp can vary the degree to which the muscle is stretched before stimulation. Frog Ringers solution (a substitute for extracellular fluid).

References: Bennett, M. (2010). Lecture 3.1: Skeletal Muscle - chemical energy to mechanical work. BIOL1040 Module 3 Lectures, The University of Queensland, Australia. Campbell, N. A., J. B. Reece and N. Meyers (2009). Biology. Frenchs Forest, NSW, Pearson Education Asutralia. Hudson, N. J., N. A. Lavidis, P. T. Choy and C. E. Franklin (2005). "Effect of prolonged inactivity on skeletal motor nerve terminals during aestivation in the burrowing frog, Cyclorana alboguttata." Journal of Comparative Physiology a-Neuroethology Sensory Neural and Behavioral Physiology 191(4): 373-379. Kibedi, J. and S. Theiss (2010). Action Potentials. BIOL1040 Practical Class. The University of Queensland, Australia.